Posted on Tue, Mar 06, 2012
Congratulations to Sandeep Khurana M.B.B.S., Hema Raina M.B.B.S., M.D., Ph.D., Valeria Pappas Ph.D., Jean-Pierre Raufman M.D., and Thomas L Pallone M.D., for their recent publication:
Khurana S, Raina H, Pappas V, Raufman J-P, Pallone TL (2012) Effects of Deoxycholylglycine, a Conjugated Secondary Bile Acid, on Myogenic Tone and Agonist-Induced Contraction in Rat Resistance Arteries. PLoS ONE 7(2): e32006. doi:10.1371/journal.pone.0032006
Background
Bile acids (BAs) regulate cardiovascular function via diverse mechanisms. Although in both health and disease serum glycine-conjugated BAs are more abundant than taurine-conjugated BAs, their effects on myogenic tone (MT), a key determinant of systemic vascular resistance (SVR), have not been examined.
doi:info:doi/10.1371/journal.pone.0032006.g005
Methodology/Principal Findings
Fourth-order mesenteric arteries (170–250 µm) isolated from Sprague-Dawley rats were pressurized at 70 mmHg and allowed to develop spontaneous constriction, i.e., MT. Deoxycholylglycine (DCG; 0.1–100 µM), a glycine-conjugated major secondary BA, induced reversible, concentration-dependent reduction of MT that was similar in endothelium-intact and -denuded arteries. DCG reduced the myogenic response to stepwise increase in pressure (20 to 100 mmHg). Neither atropine nor the combination of L-NAME (a NOS inhibitor) plus indomethacin altered DCG-mediated reduction of MT. K+ channel blockade with glibenclamide (KATP), 4-aminopyradine (KV), BaCl2 (KIR) or tetraethylammonium (TEA, KCa) were also ineffective. In Fluo-2-loaded arteries, DCG markedly reduced vascular smooth muscle cell (VSM) Ca2+ fluorescence (~50%). In arteries incubated with DCG, physiological salt solution (PSS) with high Ca2+ (4 mM) restored myogenic response. DCG reduced vascular tone and VSM cytoplasmic Ca2+ responses (~50%) of phenylephrine (PE)- and Ang II-treated arteries, but did not affect KCl-induced vasoconstriction.
doi:info:doi/10.1371/journal.pone.0032006.g005
Conclusion
In rat mesenteric resistance arteries DCG reduces pressure- and agonist-induced vasoconstriction and VSM cytoplasmic Ca2+ responses, independent of muscarinic receptor, NO or K+ channel activation. We conclude that BAs alter vasomotor responses, an effect favoring reduced SVR. These findings are likely pertinent to vascular dysfunction in cirrhosis and other conditions associated with elevated serum BAs.
doi:info:doi/10.1371/journal.pone.0032006.g005
Calcium fluorescence
In experiments where a Ca2+ indicator was used, the selected artery was exposed to dissection solution containing Fluo-2 (TEFLabs, 7.5 µM), 1.5% DMSO (vol/vol), and 0.03% cremophor EL (vol/vol) for 1 h at room temperature. After 1 h, the arteries were cannulated as described above and allowed to develop MT for 30 min. Arteries were imaged with a confocal scanning inverted microscope (×60, 1.4 NA, water-immersion objective). Images of fluo-2-loaded fluorescent VSM were obtained with an intensified CCD camera (Stanford Photonics, Palo Alto, CA, USA) coupled to a Nipkow spinning disk confocal microscope with 488 nm excitation. Spatially resolved information on cytoplasmic [Ca2+] was obtained in individual VSM cells. To quantify changes, fluo-2 fluorescence (F) was normalized to its initial value (F0) in each cell.
doi:info:doi/10.1371/journal.pone.0032006.g005
Figure 5. DCG reduces VSM Ca2+ in 4th-order mesenteric arteries from rats with MT.
Ca2+ fluorescence was measured in Fluo-2-loaded arteries before and after incubating with DCG 100 µM for 5 minutes. DCG reduced the arterial VSM Ca2+ fluorescence by ~50%. (n = 3 arteries in each group).
doi:info:doi/10.1371/journal.pone.0032006.g005
Copyright Khurana et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Effects of Deoxycholylglycine, a Conjugated Secondary Bile Acid, on Myogenic Tone and Agonist-Induced Contraction in Rat Resistance Arteries
Posted on Tue, Oct 18, 2011
A preliminary response of Asante Natrium Green-2 with sodium channel receptor, Nav 1.3 expressed in HEK 293 cells is shown in Figure 1 below. The wavelength of excitation used was 488 nm. Figure 2 shows various excitation wavelengths including emission maxima 517 nm. Filters or Flexstation may be neccessary for maximum response.
Figure 1: Na+ flux in NaV 1.3-expressing HEK-293 cells loaded with ANG-2 and treated with a concentration series of lidocaine. Cells stimulated with 25 mM vertradine in 20 mM HEPES-buffered HBSS.
Figure 2 (below): The maximum emission reached on ANG-2TMA+ at different excitation Wavelengths.
Posted on Wed, Jul 20, 2011
Rhod-2, with fluorescence excitation maxima at 552 nm and emission maxima at 581 nm, was first introduced in 1989. Rhod-2 is essentially non-fluorescent before Ca2+ binding, becoming more fluorescent with increasing Ca2+ concentration. The longer excitations and emissions of Rhod-2 make the indicator useful for experiments in cells and tissues that have high levels of autofluorescence and for multiplexing with other fluorescent dyes of shorter wavelengths.
TEFLabs offers high Quality Rhod-2 (>90% by HPLC) at significant savings compared to other Rhod-2 suppliers:
Learn more about TEFLabs Rhod-2 pricing and availability
Rhod-2 pricing (current as of 20 july 2011):
TEFLabs 1mg $110
Invitrogen 1mg $ 319
Anaspec 1mg $247.50
Enzo 1mg $239
AAT 1mg $175
Biotium 1mg $138
Learn more about TEFLabs Rhod-2 pricing and availability
Posted on Wed, Jul 13, 2011
A recent article by Christophe Lamy, and Jean-Yves Chatton highlights the uses and improved performance of TEFLabs' novel sodium indicator, Asante NaTRIUM Green-1™, over previous fluorescent sodium indicators like Sodium Green™, CoroNa Green™, and CoroNa Red™.
Sodium Green™, CoroNa Green™, and CoroNa Red™ are registered trademarks of Life Technologies, Inc.
Posted on Mon, Jun 20, 2011
TEFlabs has the lowest prices ever for Fluo-8. Beat the heat this summer, spend more time indoors performing experiments using Fluo-8. (expires 10-1-2011)
#0201 TEFlabs Fluo-8 (AM) 1 mg $50
#0203 TEFlabs Fluo-8 (AM) 20 x 50 ug $60
#0205 TEFlabs Fluo-8 (K+Salt) 1 mg $50
While Fluo-8 is marketed under a variety of names, the original patent outlining Fluo-2 medium affinity (Fluo-8) published in 1991. The original journal publication appeared in the Journal of Biological Chemistry in 1989. Why pay a premium for 20 year old indicator technology?
Some companies will charge $245 or more per mg, TEFLabs has been making Fluo dyes since 1988 and has more Fluo experience than any of our competitors, our experience and expertise at manufacturing Fluo dyes allows us to offer Fluo-8 at much lower cost to researchers. Dont let other manufacturers inexperience cost you money!
Read the original Fluo paper:
Fluorescent Indicators for Cytosolic Calcium based on Rhodamine and Fluorescein Chromophores
Posted on Mon, Jun 13, 2011
TEFLabs Inventory Reserve program allows customers to get favorable per unit pricing based on anticipated annual consumption. TEFLabs will hold a specified amount of inventory for customers who can then order throughout the year from their reserved inventory. Customers get the price benefit of a one time bulk order but only pay for products as they are used.
In addition to the best possible volume discounts the Inventory Reserve program allows researchers to receive all their reserved inventory from a single lot, guarantees the immediate availability of inventory, and allows researchers to pay for products as they are ordered without having to purchase up front and store in-house.
TEFLabs Inventory Reserve program can be used for annual orders of 10 units up to multi gram scale. Inventory Reserve orders typically receive higher bulk rate discounts than TEFlabs advertised quantity discounts.
To request an Inventory Reserve quote simply complete and return the form below:
TEFLabs Inventory Reserve Order Form
Posted on Mon, Apr 11, 2011
At the 17th annual Society of Biomolecular Screening (SBS) conference and exhibition, March 27-31 2011 in Orlando Florida, TEFLabs announced Asante NaTRIUM Green and Asante Potassium Green ion channel dyes to over 1600 scientists from 28 countries.
TEFLabs' new products were one of three winners of the first ever, SLAS New Product Award (NPA) Designations at SBS 2011, which recognized the best of what was new on the exhibit floor. NPA winners were judged by a diverse panel of scientific specialists. The SBS 2011 NPA winners included:
• InSphero: GravityPLUS System
• Pharmadiagnostics: SoPRano Surface Plasmon Resonance (SPR)-Based Screening using a Standard Absorbance Plate Reader
• Teflabs: Asante NaTRIUM Green and Asante Potassium Green Ion Channel Dyes
Posted on Mon, Jan 31, 2011
Three of the most popular long wavelength fluorescent calcium indicators; Fluo-2, Fluo-3, and Fluo-4; were invented by TEF Labs’ creator Akwasi Minta and by Nobel Laureate Roger Tsien at University of California Berkeley (US Patent 5049673, 1991).
Initially Fluo-3 was commercialized because of its low pKa. Fluo-4 became increasingly popular because of its greater "brightness" when excited at the 488 nm argon laser line. (Both Fluo-3 and Fluo-4 are approximately the same "brightness" when excited at their longer wavelength excitation maxima)
At 488 nm, Fluo-2 is the brightest of all, up to twice as bright as Fluo-4,most likely because its AM ester loads more readily into the cell. For their own reasons, other companies have renamed Fluo-2, for example as Fluo-8. In seeking transparency with our customers, we prefer to follow the original nomenclature: Fluo-2.
An experimental comparison of Fluo-2,3,4,and 8 can be found here.
Benefits of Fluo-2 MA:
- A direct substitute for Fluo-3 and Fluo-4
- The same molecule as Fluo-8
- Up to twice as bright as Fluo-4
- Up to four times as bright as Fluo-3
- AM ester loads well at room temperature, faster than Fluo-3 and Fluo-4
- Excitation at 490 nm, emission at 515 nm, and 390 nM Kd
- Greater than two hundred-fold increase from zero to saturated calcium
Posted on Thu, Jan 20, 2011
FLUO-2 MA (AM), US Patent # 5,049,673
(FLUO-2 Medium Affinity, Acetoxymethyl ester)
Fluo-2 Medium Affinity emerges as an ideal candidate over Fluo-3 and Fluo-4 for calcium study. With improved loading characteristics and brightness (nearly 2x as bright as Fluo-4, 4 x as bright as Fluo-3) Fluo-2 MA (the same molecule as Fluo-8) has become a very cost effective replacement or screeners and researchers currently using Fluo-3, Fluo-4 or Fluo-8
Fluo-2 MA, Fluo-3, Fluo-4, Fluo-8 Comparison
REF52 rat embryo fibroblasts
Procedure:
REF52 rat embryo fibroblasts, cultured on No. glass coverslips, were incubated for 45 minutes at room temperature with 5 μM of the AM ester of each indicator in serum-free DMEM containing 25 mM HEPES (pH 7.4). The coverslips were then rinsed in HBSS, mounted in round dishes and bathed with HBSS. Indicators were excited with 488-nm light from a monochromator; fluorescence images were acquired with a cooled CCD camera through a ×40 objective with high numerical aperture (NA = 1.4). Cells were stimulated with 2 μM ionomycin to elicit a sharp rise in intracellular Ca2+ concentration. The images acquired immediately before and after ionomycin addition are shown. The fluorescence change resulting
from ionomycin application in each population of cells was also analyzed; the results are displayed in the bar graph. The result for each indicator is derived from analyzing 35 – 45 cells.
Fluo-2 Products currently available:
Fluo-2 MA (Medium Affinity): Excitation at 490 nm, emission at 515 nm, and 390 nM Kd
TRIAL SIZE #0214 Fluo-2 MA (AM) 2 x 50 ug $15
TRIAL SIZE #0216 Fluo-2 MA (K+SALT) 2 x 50 ug $15
#0200 Fluo-2 MA (AM) 1 mg $110
#0202 Fluo-2 MA (AM) 20 x 50 ug $130
#0204 Fluo-2 MA (K+SALT) 1 mg $110
#0220 Fluo-2 HA (AM) 1mg $110
#0222 Fluo-2 HA (AM) 2 x 50 ug $130
#0224 Fluo-2 HA (K+SALT 1mg $110
#0240 Fluo-2 LA (AM) 1mg $130
#0242 Fluo-2 LA (AM) 2 x 50 ug $150
#0244 Fluo-2 LA (K+SALT) 1mg $130
#0230 Fluo-2 LeakRes (AM) 1mg $130
#0232 Fluo-2 LeakRes (AM) 2 x 50 ug $150
#0234 Fluo-2 LeakRes (K+SALT) 1mg $130
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Bulk discounts available, email probes@teflabs.com for bulk quotes.
Posted on Tue, Jan 11, 2011
A photomicrograph of cultured embryonic rat hippocampal neurons after 10 days in vitro. The cells were loaded with 3 uM of Asante Calcium Red AM for 30 minutes in culture media then washed in dye-free media for 30 minutes.
Cells were perfused in a flow through chamber and imaged on the stage of an inverted Nikon microscope with a 40 x 1.35 NA objective. Cells were illuminated with Xenon bulb using a 35 nm wide band of light centered around 572 nm and a 60 nm wide band of light centered around 632 nm was imaged with a cooled CCD camera. The scale bar is 20 um.
The below graph shows the percent change in Asante Red Calcium fluorescence in the cell bodies of selected neurons in response to stimulation with NMDA (300 uM) in glycine-containing, Mg-free saline. Images were obtained every 20 seconds. Regions of interest were drawn around the somata, and mean intensities within the regions recorded as a function of time. Percent change of fluorescence at each time was calculated with respect to an average of the first five images.
Images, Graph, and description provided courtesy of J Marks, University of Chicago.